CN105136946B - 12 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco leaf - Google Patents
12 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco leaf Download PDFInfo
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Abstract
The invention discloses 12 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco leaf, including the preparation of sample pre-treatments, need testing solution, the preparation of reference substance solution and determination step.The present invention identifies 17 kinds of flavonoid substances based on inventor's leaf from tobacco in 2014 and in spending, wherein 9 kinds be tobacco in first be accredited out, so that establishing the wherein 12 kinds quantitative analysis methods of flavonoid substances, the correlative study of the foundation for carrying out flavonoid substances in tobacco petal of the inventive method is significant.
Description
Technical field
The invention belongs to technical field of analysis and detection, and in particular to 12 kinds of flavonoid substances in a kind of quantitative analysis tobacco leaf
Detection method.
Background technology
Flavonoid substances (flavonoids) refer to that two phenyl ring pass through a series ofization that central thricarbon atom is connected with each other
Compound, they are distributed widely in plant kingdom, are the important secondary metabolites of plant.Flavonoid substances are in many plants
In be proved to pest-resistant and disease-resistant related to plant.Meanwhile, flavone compound is the very strong compound of a class bioactivity, is
A kind of natural antioxidant, has a wide range of applications in fields such as medicine, food.Flavonoids in tobacco（Including class
Flavone glycoside and its aglycon）Or the aroma quality and perfume quantity of the important as precursors of tobacco flavor material, its catabolite and tobacco
It is relevant.In modulation, alcoholization and combustion process in tobacco leaf, the oxidable decomposition of flavone compound assigns graceful faint scent, increases
Perfuming tolerance, therefore, they play the role of important to the quality for improving tobacco product.
At present, the analyzing detecting method report on flavonoid substances in tobacco is less.Nineteen sixty-five, Watanabe and
Wender isolates rutin sophorin, kaempferia galamga phenolic glycoside, different using the method for solvent extraction and Paper chromatography from the stamen of tobacco
The flavone compounds such as quercitin, astragalin, Quercetin -7-O- glucosides, nicotiflorin.1992, Snook etc. used liquid phase
Chromatography system have studied 66 and grow tobacco the Flavonoid substances in spending, it is found that the different middle Flavonoid substances of tobacco are widely different.
2007, Tao Pang etc. were separated from tobacco leaf using Liquid Chromatography-Tandem Mass Spectrometry and are identified rutin sophorin, Kaempferia galanga phenolic glycoside, quercitin
Deng compound.At present, the quantitative analysis method of flavonoid substances pertains only to content highest rutin sophorin in tobacco（Quercetin -3-
O- rutinosides）And FNS.This is mainly due to traditional relatively low nothing of high performance liquid chromatography sensitivity
Method detects low content flavonoid substances, and it is also one of reason not to be accredited before the flavonoid substances of part.As people are to cigarette
The increase of flavonoid substances concern in grass, setting up a kind of comprehensive quantitative analysis method of flavonoid substances becomes very necessary.
The content of the invention
It is an object of the invention to provide 12 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco leaf.
The object of the present invention is achieved like this, including the preparation of sample pre-treatments, need testing solution, reference substance solution
Prepare and determination step, specifically include：
A, sample pre-treatments：Saved backup being placed in 1 ~ 4 DEG C after the grinding of measuring samples tobacco leaf, freeze-drying；
The preparation of B, need testing solution：Sample after the accurate pre-treatment for weighing 10.0mg, add the methanol-chloroform of 1ml-
The inner mark solution of water extraction solution and 200 μ l, inner mark solution is the aqueous solution of Daidzein, genistin and aurantiamarin, concentration
It is 1.0 μ g/mL, supernatant is taken as need testing solution after sonicated, centrifugation；
The preparation of C, reference substance solution：The accurately weighed each n-compound of difference, i.e., 12 standard samples and 3 internal standard compounds
Daidzein, genistin and aurantiamarin, plus methyl alcohol are made solution of every 1ml containing 1mg, and the flavonoids list mark for obtaining 1mg/ml is molten
Liquid, pipette respectively 100 μ l 1mg/ml low concentration flavonoids singly mark solution and 10mg/ml high concentration flavonoids list mark it is molten
It is molten that liquid prepares standard mixing in 100ml volumetric flasks, with sample extraction solution, i.e. methanol-chloroform-water extraction solution constant volume
Liquid, with sample extraction solution stepwise dilution standard mixed solution, be made low concentration flavonoid substances concentration for 1000,600,400,
100th, 60,40,10,6,4,1ng/mL, volume is the standard liquid gradient of 1mL；The corresponding high standard solution ladder of this gradient
It is 100,60,40,10,6,4,1,0.6,0.4,0.1 μ g/mL to spend；Add 200 μ l's in the solution of above-mentioned all concentration gradients
Inner mark solution obtains reference substance solution, and inner mark solution is the aqueous solution of Daidzein, genistin and aurantiamarin, and concentration is 1.0 μ
g/mL；
D, measure：Through chromatograph mass spectrum analysis, the content with 12 flavonoids to be measured is corresponding with its as abscissa
Chromatographic peak area is ordinate with the ratio of the chromatographic peak area of internal standard compound, sets up working curve, and correction data is carried out
Linear regression, tries to achieve the regression equation of flavonoid substances, and the content of flavonoid substances is calculated as the following formula in sample：
In formula：C is the content of flavonoid substances in tobacco petal sample, unit μ g/g；
The concentration of the extract that x is measured for instrument, unit is μ g/mL.
The present invention identifies 17 kinds of flavonoid substances based on inventor's leaf from tobacco in 2014 and in spending, wherein 9 kinds are
It is accredited out first in tobacco so that establish the wherein 12 kinds quantitative analysis methods of flavonoid substances, the inventive method
The correlative study set up for carrying out flavonoid substances in tobacco petal is significant.
Brief description of the drawings
Fig. 1 is the flavonoid substances separation chromatography figure of tobacco sample of the present invention；
In figure：The corresponding compound of chromatogram of 1. ~ numbering is followed successively by Quercetin -3-O- rutin sophorins, Quercetin -3-O- Portugals
Polyglycoside, Kaempferol -3-O- rutin sophorins, Isorhamnetin -3-O- rutinosides, Kaempferol -3-O- glucosides, Isorhamnetin -
3-O- glucosides, dihydroquercetin, naringenin -7-O- glucose, dihydrokaempferol, Luteolin, Quercetin, different sandlwood
Element；
Fig. 2 is that the flavonoid substances of tobacco sample of the present invention separate TIC.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is further illustrated, but the present invention is subject to never in any form
Limitation, based on present invention teach that any conversion or replacement made, belong to protection scope of the present invention.
12 kinds of detection methods of flavonoid substances in quantitative analysis tobacco leaf of the present invention, including sample pre-treatments, confession
The preparation of test sample solution, the preparation of reference substance solution and determination step, specifically include：
A, sample pre-treatments：Saved backup being placed in 1 ~ 4 DEG C after the grinding of measuring samples tobacco leaf, freeze-drying；
The preparation of B, need testing solution：Sample after the accurate pre-treatment for weighing 10.0mg, add the methanol-chloroform of 1ml-
The inner mark solution of water extraction solution and 200 μ l, inner mark solution is the aqueous solution of Daidzein, genistin and aurantiamarin, concentration
It is 1.0 μ g/mL, supernatant is taken as need testing solution after sonicated, centrifugation；
The preparation of C, reference substance solution：The accurately weighed each n-compound of difference, i.e., 12 standard samples and 3 internal standard compounds
Daidzein, genistin and aurantiamarin, plus methyl alcohol are made solution of every 1ml containing 1mg, and the flavonoids list mark for obtaining 1mg/ml is molten
Liquid, pipette respectively 100 μ l 1mg/ml low concentration flavonoids singly mark solution and 10mg/ml high concentration flavonoids list mark it is molten
It is molten that liquid prepares standard mixing in 100ml volumetric flasks, with sample extraction solution, i.e. methanol-chloroform-water extraction solution constant volume
Liquid, with sample extraction solution stepwise dilution standard mixed solution, be made low concentration flavonoid substances concentration for 1000,600,400,
100th, 60,40,10,6,4,1ng/mL, volume is the standard liquid gradient of 1mL；The corresponding high standard solution ladder of this gradient
It is 100,60,40,10,6,4,1,0.6,0.4,0.1 μ g/mL to spend；Add 200 μ l's in the solution of above-mentioned all concentration gradients
Inner mark solution obtains reference substance solution, and inner mark solution is the aqueous solution of Daidzein, genistin and aurantiamarin, and concentration is 1.0 μ
g/mL；
D, measure：Through chromatograph mass spectrum analysis, the content with 12 flavonoids to be measured is corresponding with its as abscissa
Chromatographic peak area is ordinate with the ratio of the chromatographic peak area of internal standard compound, sets up working curve, and correction data is carried out
Linear regression, tries to achieve the regression equation of flavonoid substances, and the content of flavonoid substances is calculated as the following formula in sample：
In formula：C is the content of flavonoid substances in tobacco petal sample, unit μ g/g；
The concentration of the extract that x is measured for instrument, unit is μ g/mL.
Grinding described in step A is that measuring samples tobacco leaf is put into mortar, and liquid feeding chilled nitrogen grinds.
The volume proportion of the methanol-chloroform-water extraction solution described in step A is 5:2:2.
Chromatographiccondition described in D steps is：Chromatographic column, Waters BEH C18（15cm × 2.1mm, 1.7 μm of grains
Footpath）；A phases, water；B phases, acetonitrile, two-phase adds 0.1% formic acid and the ammonium acetate of 0.2mmol/L；Mobility gradient, 0-
1min, 10%B；1-9min, 10%B-90%B；9-11min, 90%B-100%B；11-11.1min, 100%B-10%B；11.1-
13min, keeps 10%B；30 DEG C of column temperature, sample size 2 μ L, flow velocity 0.25mL/min.
Mass spectral analysis condition described in D steps is：Ion gun, electron spray ionisation ion gun；Spray voltage, -4000V；
Curtain atmospheric pressure, 40psi；Ion source temperature, 700 DEG C；Auxiliary gas 1,60psi；Auxiliary gas 2,50psi；Remove cluster voltage, -100V；Change
Compound quota ion and energy are shown in Table 1,
The flavonoid substances of table 1 analyze mass spectrum quota ion and collision energy
With embodiment, the invention will be further described below：
Experiment reagent and device：
Acetonitrile（Chromatographic grade）, methyl alcohol（Chromatographic grade）, ethanol（Chromatographic grade）Buy in German Merck companies.Ultra-pure water by
It is prepared by Millipore purification systems.Dihydrokaempferol, dihydroquercetin, Quercetin, rutin sophorin, Kaempferol -3-O- rutinoses
Glycosides, Isorhamnetin, Quercitrin-3-O-glucoside, cyanidenon, Isorhamnetin -3-O- glucosides, Isorhamnetin -3-O-
Standard substance and Daidzein, the dyewoods such as rutinoside, Kaempferol -3-O- glucosides, eriodictyol-7- O -glucoside
Glycosides（Glucosyl group）, aurantiamarin（Rue glycosyl）Bought in Sigma-Aldrich, Alfa Aesar and lark etc. internal standard substance
Prestige company.Waters UPLC liquid chromatographs（The U.S.）, the triple quadrupole mass spectrometers of AB SCIEX 5500（The U.S.).SB-50D
Ultrasonic wave extraction instrument（NingBo XinZhi Biology Science Co., Ltd）；Waters BEH C18（15cm×2.1 mm, 1.7μm
Particle diameter）Reverse-phase chromatographic column（Waters, USA）, MILLI-Q water purification machines（MILLIPORE companies）, LD5-2A centrifuges（System in Beijing Jing
Vertical centrifuge Co., Ltd）, vortex vortex mixer（Dutch Breda companies）；CP2245 assay balances（Sensibility reciprocal 0.0001g, Germany
Sartorious companies）.
Embodiment 1
--- the quantitative analysis of tobacco leaf flavonoid substances in tobacco cultivars K326
Experiment material：Fresh lyophilized K326 maturity periods middle part tobacco leaf.
Experimental technique：
The accurate lyophilized tobacco leaf sample for weighing 10.0 mg, adds 1mL extractants（Methanol-chloroform-water, 5:2:2, volume
Than）With 200 μ L inner mark solutions（1.0μg/mL）, ultrasonic 30min, centrifugation takes supernatant and is transferred to liquid chromatogram sample introduction bottle point
Analysis.
Chromatographiccondition：Chromatographic column, Waters BEH C18（15 cm × 2.1mm, 1.7 μm of particle diameters）；A phases, water；B
Phase, acetonitrile, two-phase adds 0.1% formic acid and the ammonium acetate of 0.2 mmol/L；Mobility gradient, 0-1min, 10%B；1-
9min, 10%B-90%B；9-11min, 90%B-100%B；11-11.1min, 100%B-10%B；11.1-13min, keeps 10%B；
30 DEG C of column temperature, sample size 2 μ L, flow velocity 0.25mL/min.
Mass spectral analysis condition：Ion gun, electron spray ionisation ion gun；Spray voltage, -4000V；Curtain atmospheric pressure, 40psi；
Ion source temperature, 700 DEG C；Auxiliary gas 1,60psi；Auxiliary gas 2,50psi；Remove cluster voltage, -100V.Compound quota ion and
Energy is shown in Table 1,
Prepare all n-compounds（It is shown in Table 1,12 standard specimens and 3 internal standards）1mg/mL singly mark solution（Methyl alcohol is
Solvent）.The low concentration flavonoids singly mark solution of 100 μ L 1mg/mL and the high concentration flavonoids of 10mL 1mg/mL are pipetted respectively
Singly mark solution in 100mL volumetric flasks, with sample extraction solution (methanol-chloroform-water, 5:2:2, volume ratio) constant volume, it is made standard
Mixed solution.With sample extraction solution stepwise dilution standard mixed solution, be made low concentration flavonoid substances concentration for 1000,
600th, 400,100,60,40,10,6,4,1ng/mL, volume is the standard liquid gradient of 1mL.The corresponding highly concentrated scale of this gradient
Quasi- solution gradient is 100,60,40,10,6,4,1,0.6,0.4,0.1 μ g/mL.200 are added in the solution of all concentration gradients
μ L internal standard mixed solutions.Internal standard mixed liquor is Daidzein, genistin, the aqueous solution of aurantiamarin, and concentration is 1.0 μ g/mL.
Daidzein, genistin, aurantiamarin are respectively used to correction sugar-free base class flavone aglycone, glucosyl group substitution flavonoids and rue
Glycosyl replaces flavonoids.Under the conditions of selected chromatographic mass spectrometry, the content with 12 flavonoids to be measured as abscissa, with
Its corresponding chromatographic peak area is ordinate with the ratio of the chromatographic peak area of internal standard compound, sets up working curve.To correction
Data carry out linear regression, try to achieve the regression equation of flavonoid substances.The content of flavonoid substances, is counted as the following formula in sample
Calculate：
In formula：C is the content of flavonoid substances in tobacco petal sample, unit μ g/g；
The concentration of the extract that x is measured for instrument, unit is μ g/mL.
Experimental result：
The content of tobacco leaf flavonoid substances in the tobacco cultivars K326 of table 2
Embodiment 2
--- the quantitative analysis of tobacco leaf flavonoid substances in makhorka N. rustica
Experiment material：Fresh lyophilized N. rustica maturity periods middle part tobacco leaf.
Experimental technique：
The accurate lyophilized tobacco leaf sample for weighing 10.0mg, adds 1mL extractants（Methanol-chloroform-water, 5:2:2, volume
Than）With 200 μ L inner mark solutions（1.0μg/mL）, ultrasonic 30min, centrifugation, take supernatant be transferred to liquid chromatogram sample introduction bottle analysis.
Chromatographiccondition：Chromatographic column, Waters BEH C18（15cm × 2.1 mm, 1.7 μm of particle diameters）；A phases, water；B
Phase, acetonitrile, two-phase adds 0.1% formic acid and the ammonium acetate of 0.2mmol/L；Mobility gradient, 0-1min, 10%B；1-
9min, 10%B-90%B；9-11min, 90%B-100%B；11-11.1min, 100%B-10%B；11.1-13min, keeps 10%B；
30 DEG C of column temperature, sample size 2 μ L, flow velocity 0.25mL/min.
Mass spectral analysis condition：Ion gun, electron spray ionisation ion gun；Spray voltage, -4000V；Curtain atmospheric pressure, 40psi；
Ion source temperature, 700 DEG C；Auxiliary gas 1,60psi；Auxiliary gas 2,50psi；Remove cluster voltage, -100V.Compound quota ion and
Energy is shown in Table 1；
Prepare all n-compounds（It is shown in Table 1,12 standard specimens and 3 internal standards）1mg/mL singly mark solution（Methyl alcohol is
Solvent）.The low concentration flavonoids singly mark solution of 100 μ L 1mg/mL and the high concentration class Huang of the mg/mL of 10mL 1 are pipetted respectively
Ketone singly mark solution in 100mL volumetric flasks, with sample extraction solution (methanol-chloroform-water, 5:2:2, volume ratio) constant volume, it is made mark
Quasi- mixed solution.With sample extraction solution stepwise dilution standard mixed solution, be made low concentration flavonoid substances concentration for 1000,
600th, 400,100,60,40,10,6,4,1ng/mL, volume is the standard liquid gradient of 1 mL.The corresponding highly concentrated scale of this gradient
Quasi- solution gradient is 100,60,40,10,6,4,1,0.6,0.4,0.1 μ g/mL.200 are added in the solution of all concentration gradients
μ L internal standard mixed solutions.Internal standard mixed liquor is Daidzein, genistin, the aqueous solution of aurantiamarin, and concentration is 1.0 μ g/mL.It is yellow
Beans aglycon, genistin, aurantiamarin are respectively used to correction sugar-free base class flavone aglycone, glucosyl group substitution flavonoids and rutinose
Base replaces flavonoids.Under the conditions of selected chromatographic mass spectrometry, the content with 12 flavonoids to be measured as abscissa, with it
Corresponding chromatographic peak area is ordinate with the ratio of the chromatographic peak area of internal standard compound, sets up working curve.To correction number
According to linear regression is carried out, the regression equation of flavonoid substances is tried to achieve.The content of flavonoid substances, is counted as the following formula in sample
Calculate：
In formula：C is the content of flavonoid substances in tobacco petal sample, unit μ g/g；
The concentration of the extract that x is measured for instrument, unit is μ g/mL.
Experimental result：
The content of tobacco leaf flavonoid substances in the makhorka N. rustica of table 3
Test example 1
Specification Curve of Increasing is carried out according to described method, the linearly dependent coefficient of all compounds is found（r 2 ）Exist
More than 0.99, illustrate that set up method has good linear response, it is adapted to quantitative analysis.These compounds are quantified
Limit and test limit assessment, it is found that test limit and the quantitative limit difference of different compounds are larger, wherein Isorhamnetin -3- glucose
Glycosides, dihydroquercetin, naringenin -7- glucose, dihydrokaempferol, Isorhamnetin -3-O- rutinosides, Kaempferol -3-O- Portugals
Polyglycoside, the quantitative limit of Quercitrin-3-O-glucoside can reach 1.2~5.6ng/mL, and Quercetin -3-O- rutin sophorins, mountain
How the quantitative limit of phenol -3-O- rutin sophorins, cyanidenon, Quercetin, Isorhamnetin etc. is between 0.065~0.4 μ g/mL（It is shown in Table
4）.Flavonoid substances are carried out with repeated investigation, finds the in a few days repeatability of investigated flavonoid substances 3.5%~7.4%
Between, repeatability is between 5.2%~11.4% in the daytime（It is shown in Table 5）.According to 11 kinds of cigarettes that flavonoid substances are determined in this experiment
Mean concentration in grass, adds the assay method rate of recovery in standard sample to the actual sample of mean concentration about 50% and 100%, knot
Fruit finds the rate of recovery of this method between 86.3%~108.6%（It is shown in Table 5）.
The calibration curve of the flavonoids of table 4, test limit and quantitative limit
The repeatability and the rate of recovery of the flavonoids of table 5
Claims (2)
1. 12 kinds of detection methods of flavonoid substances in a kind of quantitative analysis tobacco leaf, it is characterised in that including sample pre-treatments, supply
The preparation of test sample solution, the preparation of reference substance solution and determination step, specifically include：
A, sample pre-treatments：Saved backup being placed in 1 ~ 4 DEG C after the grinding of measuring samples tobacco leaf, freeze-drying；
The preparation of B, need testing solution：Sample after the accurate pre-treatment for weighing 10.0mg, it is 5 to add 1mL volume proportions:2:2
Methanol-chloroform-water extraction solution and 200 μ L inner mark solution, inner mark solution is for Daidzein, genistin and aurantiamarin
The aqueous solution, concentration be 1.0 μ g/mL, sonicated 30min, centrifugation after take supernatant as need testing solution；
The preparation of C, reference substance solution：The accurately weighed each n-compound of difference, i.e., 12 standard items, plus methyl alcohol is made every 1mL
Solution containing 1mg, obtains the flavonoids singly mark solution of 1mg/mL, pipette respectively the flavonoids singly mark solution of 100 μ L 1mg/mL with
And the flavonoids singly mark solution of 10mL 1mg/mL is in 100mL volumetric flasks, with sample extraction solution, i.e. methanol-chloroform-water extraction
Take solution constant volume and prepare standard mixed solution, with sample extraction solution stepwise dilution standard mixed solution, be made flavonoids
Material concentration is 1000,600,400,100,60,40,10,6,4, the low concentration standard liquid gradient of 1ng/mL；This gradient correspondence
High standard solution gradient be 100,60,40,10,6,4,1,0.6,0.4,0.1 μ g/mL；In above-mentioned all concentration gradients
Solution in add the inner mark solution of 200 μ L to obtain reference substance solution, inner mark solution is Daidzein, genistin and aurantiamarin
The aqueous solution, concentration be 1.0 μ g/mL；
D, measure：Through chromatograph mass spectrum analysis, the content with 12 flavonoids to be measured as abscissa, with its corresponding chromatogram
Peak area is ordinate with the ratio of the chromatographic peak area of internal standard compound, sets up working curve, and correction data is carried out linearly
Return, try to achieve the regression equation of flavonoid substances, the content of flavonoid substances is calculated as the following formula in sample：
In formula：It is the content of flavonoid substances in tobacco sample, unit μ g/g；
X is the concentration of need testing solution, and unit is μ g/mL；
Chromatographiccondition is：Chromatographic column, Waters BEH C18 15cm × 2.1mm, 1.7 μm of particle diameters；A phases, water；B phases, second
Nitrile, two-phase adds 0.1% formic acid and the ammonium acetate of 0.2mmol/L；Mobility gradient, 0-1min, 10%B；1-9min, 10%
B-90%B；9-11min, 90%B-100%B；11-11.1min, 100%B-10%B；11.1-13min, keeps 10%B；30 DEG C of column temperature,
Sample size 2 μ L, flow velocity 0.25mL/min；
Mass spectral analysis condition is：Ion gun, electron spray ionisation ion gun；Spray voltage, -4000V；Curtain atmospheric pressure, 40psi；From
Source temperature, 700 DEG C；Auxiliary gas 1,60psi；Auxiliary gas 2,50psi；Remove cluster voltage, -100V；Compound quota ion and touch
Hit energy and be shown in Table 1,
The flavonoid substances of table 1 analyze mass spectrum quota ion and collision energy
。
2. 12 kinds of detection methods of flavonoid substances in quantitative analysis tobacco leaf according to claim 1, it is characterised in that A is walked
Grinding described in rapid is that measuring samples tobacco leaf is put into mortar, and liquid feeding chilled nitrogen grinds.
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